Erthrocruorins and Chlorocruorins are large (mol. wt. 0.5. to 3 x 10 to the 6th power) invertebrate proteins and glycoproteins possessing a diverse and complex quaternary structure, capable of combining reversibly with oxygen. The objectives of the proposed research are the following: (1) the determination of the subunit structures of several annelid, molluscan and crustacean erythrocruorins and chlorocruorins; (2) the isolation of the smallest heme-binding subunit from Areni cola and Lumbricus erythrocruorins; (3) the determination of the primary structures of the isolated subunits; (4) an investigation of the biosynthesis and the assembly of the subunits of erythrocruotins in culture chloragocytes. The subunit structure of erythrocruorins will be determined using gel filtration and polyacrylamide gel electrophoresis in the presence of detergents and at alkaline pH. Cross-linking of the subunits in the intact molecule will be carried out using bifunctional reagents followed by identification of the cross-linked subunits using previously developed methods of dissociation. The isolation of the smallest heme-binding subunits will be achieved by gel filtration at alkaline pH. The primary structure of these subunits of Lumbricus and Arenicola erythrocruorins will be determined using the Edman degradation method and the peptides obtained by proteolytic digestion and chemical cleavage of the polypeptide chain. The synthesis of the polypeptide chains constituting Arenicola or Lumbricus erythrocruorin by chloragocytes growing in culture will be investigated using radioactively labeled precursors. The role of post-translational glycosylation in the assembly of the subunits will also be examined.